This project will characterize the minimal promoter region of human NAT1 and NAT2 and study effects of genetic polymorphisms on expression in human mammary cells. Specific Aim 1 involves identifying the NAT1 and NAT2 transcriptional start site in human mammary epithelial cells by 5'-RLM-RACE. Specific Aims 2 and 3 will identify the functional promoter of NAT1 and NAT2 by construction of luciferase reporter genes containing 5'-UTR regions of NAT1 and AT2 and site-directed mutagenesis and deletion analysis, and assessing the transcriptional effects of these regions by transient expression in MCF-7 and MCF-10A cell lines. Specific Aim 4 involves investigating transcriptional regulation of NAT1 and NAT2 in normal human mammary tissues by determining NAT1 and NAT2 mRNA levels, protein expression, intrinsic protein stability, and catalytic activity for N- and O-acetylation activities as a function of 5'-UTR and coding region genotypes. Specific Aim 5 involves sequencing the 5'-UTR of NAT1 and NAT2 in human mammary tissues to identify and characterize effects of polymorphisms in the 5'-UTR of NAT1 and NAT2. Knowledge of transcriptional regulation in the 5'-UTR of NAT1 and NAT2 will allow for an increased understanding the role of genetic polymorphisms towards genetic predisposition to breast cancer from environmental carcinogens.